Apoptosis-Induction by Voacanga globosa of Human Colon Carcinoma Cells
Asian Pac J Cancer Prev, 15 (2), 617-622
Introduction
During carcinogenesis, certain mutations suppress apoptosis allowing the continued survival of cancer cells (Hanahan and Weinberg, 2000; Varfolomeev and Vucic, 2011). The Department of Health has reported malignant neoplasms to be the third leading cause of mortality in the Philippines from 2004-2008 and in
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induce apoptosis. It is widely accepted that induction of apoptosis plays a role in killing cancer cells in vitro and is an attractive target for cancer therapy (Blagosklonny, 2004; Strasser et al., 2011).
In this study, leaves of Voacanga globosa were investigated for possible cytotoxicity against cancer cells.
A previous study has demonstrated that extracts from V globosa leaves exhibit high toxicity against two cancer cell lines, HCT116 human colon carcinoma and A549 human lung carcinoma (Canoy, 2011). V globosa is thus
1Mammalian Cell Culture Laboratory, Institute of Biology, 2Institute of Chemistry, College of Science, University of the Philippines, Diliman, Quezon City, Philippines *For correspondence: eternal_alvin@yahoo.com
Abstract
Voacanga globosa (Blanco), a plant endemic to the Philippines, is traditionally used especially by indigenous people of Bataan in the treatment of ulcers, wounds and tumorous growths. This study aimed to provide VFLHQWLÀF HYLGHQFH WR WKHUDSHXWLF SURSHUWLHV E\ GHWHUPLQLQJ F\WRWR[LF DQG SURDSRSWRWLF DFWLYLW\ RI +3/&
IUDFWLRQVIURPOHDYHVRQ+&7KXPDQFRORQFDUFLQRPDDQG$ KXPDQOXQJFDUFLQRPDFHOOOLQHV(WKDQROLF
H[WUDFWLRQ ZDV SHUIRUPHG RQ V globosa OHDYHV IROORZHG E\ KH[DQH DQG HWK\O DFHWDWH SDUWLWLRQLQJ 6LOLFD JHO
FROXPQ FKURPDWRJUDSK\ DQG KLJK SHUIRUPDQFH OLTXLG FKURPDWRJUDSK\ +3/& SURGXFHG 03 03 DQG
03IUDFWLRQV&\WRWR[LFDFWLYLW\RIWKHIUDFWLRQVZDVGHWHUPLQHGWKURXJK077DVVD\DJDLQVWWKHFDQFHUFHOO
OLQHV+&7DQG$ DQGWKHQRQFDQFHU$$&KLQHVHKDPVWHURYDULDQFHOOOLQH3URDSRSWRWLFDFWLYLWLHVRI
WKHPRVWDFWLYHIUDFWLRQVZHUHIXUWKHUDVVHVVHGWKURXJK'$3,VWDLQLQJ781(/DVVD\DQG-&PLWRFKRQGULDO
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fraction induced formation of apoptotic bodies, condensed DNA and other morphological changes consistent ZLWK DSRSWRVLV RI +&7 FHOOV DQG 781(/ DVVD\ VKRZHG VLJQLÀFDQW LQFUHDVH LQ '1$ IUDJPHQWDWLRQ RYHU
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associated with the beginning of apoptosis. Phytochemical analysis demonstrated the presence only of saponins DQGWHUSHQRLGVLQWKH03IUDFWLRQ7KHUHVXOWVLQGLFDWHWKDWWKH03IUDFWLRQH[HUWVF\WRWR[LFDFWLYLW\RQ
+&7FHOOVYLDLQGXFWLRQRIDSRSWRVLVWULJJHUHGE\ORVVRIPLWRFKRQGULDOPHPEUDQHSRWHQWLDOFUXFLDOIRUFHOO
survival.
Keywords: Voacanga globosa - cancer cell lines - cytotoxicity - apoptosis - saponins - terpenoids
RESEARCH ARTICLE
$SRSWRVLV,QGXFLQJ$FWLYLW\RI+3/&)UDFWLRQIURPVoacanga globosa %ODQFR 0HUURQWKH+XPDQ&RORQ&DUFLQRPD&HOO
/LQH+&7
Alvin Resultay Acebedo
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1likely to produce anti-cancer compounds. Using MTT assay, the study aimed to test HPLC fractions from the V globosa leaf extract for cytotoxicity against the HCT116 human colon carcinoma, A549 human lung carcinoma and the non-cancer AA8 Chinese hamster ovarian cell lines. Possible pro-apoptotic activity was also assessed using DAPI nuclear staining, TUNEL assay and the JC-1 mitochondrial membrane potential assay.
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Production of the ethanolic crude extract
Mature leaves from V globosa (Blanco) Merr. were KDUYHVWHGIURP0RURQJ%DWDDQ3KLOLSSLQHV,GHQWLÀFDWLRQ
ZDVYHULÀHGE\WKH-RVH9HUD6DQWRV+HUEDULXPRIWKH
Institute of Biology, University of the Philippines Diliman (a voucher specimen sample submitted was assigned the accession number: 14609). Leaves were air dried until crispy and then macerated in 95% technical grade ethanol IRUDWOHDVWKRXUV7KHOHDIVXVSHQVLRQZDVÀOWHUHGDQG
concentrated by rotary evaporation at 37°C producing the ethanolic crude extract.
Solvent partitioning and column chromatography For the next stages of extraction, all solvents used were of analytical grade. Ethanolic crude extract was partitioned with equal parts hexane. Hexane and ethanol partitions were concentrated by rotary evaporation. The ethanol fraction produced was then partitioned with equal parts ethyl acetate and distilled water. The resulting aqueous and ethyl acetate partitions were also concentrated separately by rotary evaporation. The hexane, ethyl acetate and aqueous partitions were reconstituted to ÀQDOFRQFHQWUDWLRQVRIPJP/'062WKHQWHVWHGIRU
cytotoxicity using the MTT assay.
The solvent partition with significant cytotoxic activity was further fractionated using normal silica gel column chromatography with 9:1 chloroform-methanol solvent system. Separation was monitored using thin layer chromatography (TLC) silica gel 60 F254 plates with 33% ethyl acetate-hexane solution as the mobile SKDVH )UDFWLRQV ZLWK VLPLODUEDQGLQJ SURÀOHV ZHUH UH
GLVVROYHGLQ FKORURIRUPPHWKDQROSRROHGDLUGULHG
and re-tested for cytotoxicity. The pooled fraction with the most significant cytotoxic activity was reloaded onto a silica gel column chromatography using a solvent gradient system of increasing polarity (elution starting at
HWK\O DFHWDWHKH[DQH ZLWK LQFUHDVLQJ HWK\O DFHWDWH
concentrations at 10% increments). Fractions from the gradient silica gel column were subjected to TLC.
Those with similar banding profiles were dissolved LQ FKORURIRUPPHWKDQRO UHSRROHG WKHQ WHVWHG IRU
F\WRWR[LFLW\7KHSRROHGIUDFWLRQZLWKWKHPRVWVLJQLÀFDQW
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performance liquid chromatography (HPLC).
High performance liquid chromatography (HPLC)
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+3/&JUDGHPHWKDQRO ƫ/RIWKHH[WUDFWZDVWKHQ
injected into the HPLC with a semi-preparative column XVLQJDÀOWHUHGDFHWRQLWULOH +3/&JUDGH GHLRQL]HGZDWHU
JUDGLHQWVROYHQWV\VWHP 7RWDOÁRZ P/PLQ %FRQF
B. curve: 0; P max: 25 Mpa; P. min: 0 MPa). The resulting +3/&SURÀOHZDVPRQLWRUHGXVLQJWKH&/$6693
program. Fractions corresponding to each major peak were collected, dried using rotary evaporation and then O\RSKLOL]HG'ULHGIUDFWLRQVZHUHUHGLVVROYHGLQGLPHWK\O
VXOIR[LGH '062 WRDÀQDOFRQFHQWUDWLRQRIPJP/
Phytochemical screening
Phytochemical screening was performed with PRGLÀFDWLRQV IROORZLQJ SURWRFROV RI +DUERUQH and Edeoga et al. (2005). Screening was performed for the following organic compounds: tannins, saponins, terpenoids, flavonoids, cardiac glycosides, phenolic compounds, steroids and alkaloids. At least two trials were performed for each phytochemical test.
Methyl thiol tetrazolium (MTT) cytotoxicity assay
7KH 077 SUROLIHUDWLRQF\WRWR[LFLW\ DVVD\ ZDV
SHUIRUPHGZLWKPRGLÀFDWLRQVDFFRUGLQJWRWKHSURWRFRORI
Mossman, 1983. Cells were washed, treated with 0.05%
trypsin-EDTA and seeded at 410FHOOVP/LQVWHULOH
well microtiter plates. The plates were incubated overnight at 37°C and 5% CO2. Cells were then treated with DMSO (negative control), doxorubicin (positive control), or the V globosa plant extracts. After 72 hours of incubation, the PHGLDZHUHUHSODFHGZLWKƫ/077VROXWLRQ PJP/
PBS). Plates were then re-incubated for 4 hours then added ZLWKƫ/'062WRHDFKZHOO$EVRUEDQFHVZHUHWKHQ
read using the LEDETECT reader at 570 nm. IC50 values were derived through linear regression. Three trials with at least two replicates per concentration were performed.
Diamidino-2-Phenylindole (DAPI) nucleic acid staining HCT116 cells were washed, detached with 0.05%
trypsin-EDTA and seeded into sterile 96 well plates at 4104FHOOP/&HOOVZHUHLQFXEDWHGRYHUQLJKWDW&
and 5% CO2 then treated using the IC50 concentrations HVWDEOLVKHG E\ WKH 077 DVVD\ IRU 03 ƫJP/
doxorubicin (positive control) and DMSO (negative FRQWURO 'R[RUXELFLQZDVWKHSRVLWLYHFRQWURODW ƫJ
mL and DMSO was the negative control. After 12, 24, 48 DQGKRXUVLQFXEDWLRQƫ/RI;'$3,ZDVDSSOLHG
per well then incubated at 37°C for at least 60 minutes.
&HOOV ZHUH YLHZHG XQGHU EULJKW ÀHOG DQG ÁXRUHVFHQFH
microscopy. Cells with morphologies indicative of apoptosis (breaking up into apoptotic bodies) were scored as positive. Three trials with three replicates per concentration were done with at least 200 cells scored per replicate. Apoptotic index was computed as number of FHOOVSRVLWLYHIRUDSRSWRVLVWRWDOQXPEHURIFHOOVVFRUHG
Terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay
DNA fragmentation was assayed following the manufacturer’s instructions of the APO-BrdU TUNEL DVVD\ NLW ZLWK VRPH PRGLÀFDWLRQV +&7 FHOOV ZHUH
ZDVKHGWU\SVLQL]HGDQGVHHGHGLQWRVWHULOH ZHOOSODWHV
at 4104FHOOP/WKHQLQFXEDWHGRYHUQLJKWDW&DQG
5% CO2. Test extracts were applied at IC50 concentration.
Ttreated cells were incubated at 37°C for 48, 60 and 72 KRXUVWKHQWU\SVLQL]HGDQGKDUYHVWHG&HOOVZLWKVLPLODU
treatment regimes were pooled into microfuge tubes. At least four wells per replicate for each treatment regime were pooled together. Cells were washed with PBS and À[HGZLWKFROGSDUDIRUPDOGHK\GHLQ3%6RQLFHIRU
30 minutes then with PBS and resuspended in 0.5 mL PBS with 5 mL cold 70% ethanol. The cell suspensions were then stored in ice for not less than twenty-four hours then centrifuged. The ethanol from each tube was discarded.
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DNA labeling solution at 37°C for two hours and shaken every 30 minutes. Cell suspensions were then washed ZLWKWKHULQVHEXIIHUDQGLQFXEDWHGZLWKƫ/DQWLERG\
staining solution at room temperature for 30 minutes.
Cells were viewed under fluorescence microscopy.
7KRVHGLVSOD\LQJEULJKWJUHHQÁXRUHVFHQFHZHUHVFRUHG
as positive for DNA fragmentation and apoptosis. A PLQLPXPRIÀYHWRWHQÀHOGVRIYLHZSHUUHSOLFDWHZDV
used for scoring of cells. Three trials with three replicates each concentration were performed and at least 200 cells per replicate were scored. The apoptotic index for each
Apoptosis-Induction by Voacanga globosa of Human Colon Carcinoma Cells replicate was computed as previously described for the
DAPI nucleic acid staining.
JC-1 mitochondrial membrane potential assay
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using the MitoProbe™ JC-1 Assay Kit following the PDQXIDFWXUHU·V LQVWUXFWLRQV ZLWK VRPH PRGLÀFDWLRQV
+&7FHOOVZHUHZDVKHGWU\SVLQL]HGVHHGHGLQWRVWHULOH
96-well plates at 4104FHOOP/WKHQLQFXEDWHGRYHUQLJKW
at 37°C and 5% CO2. Test extracts were then applied to the cells at their respective IC50 concentrations, followed by re-incubation at 37°C for seventy-two hours. Cells ZHUHWKHQWU\SVLQL]HGDQGFROOHFWHG$WOHDVWIRXUZHOOV
per replicate for similar treatment regime were pooled together into microfuge tubes. Cells were centrifuged and the supernatant from each tube was removed. The resulting cell pellets were re-suspended in 1.0 mL warm PBS.
JC-1 stock solution was added to each cell suspension at
ƫ/WKHQLQFXEDWHGDW&&22 for 45 minutes.
Cell suspensions were then centrifuged, washed with PBS, transferred to a 96-well plate and then viewed with ÁXRUHVFHQFHPLFURVFRS\XVLQJDSSURSULDWHÀOWHUV&HOOV
displaying green (positive for mitochondrial membrane GHSRODUL]DWLRQ DQG UHG QHJDWLYH IRU PLWRFKRQGULDO
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of cells. Three trials with three replicates each were performed and at least 200 cells per replicate were scored.
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Statistical analysis
Normality of distribution and homogeneity of the variances of means were assessed through the Shapiro- Wilk test and the Levene’s test respectively. Means ZHUHWKHQDQDO\]HGZLWK2QHZD\$QDO\VLVRI9DULDQFH
(ANOVA). The results of ANOVA from data sets with XQHTXDO YDULDQFHV RI PHDQV ZHUH FRQÀUPHG XVLQJ WKH
Brown-Forsythe test. Post hoc tests (Games-Howell test
for data sets with unequal variance and Tukey’s Honestly 6LJQLÀFDQWWHVWIRUGDWDVHWVZLWKHTXDOYDULDQFH ZHUHWKHQ
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means. P-values less than 0.05 indicates significant differences between means. Three independent trials with at least two replicates each were performed for each experiment. All statistical analysis were performed using the SPSS Statistics 17.0 software.
Results
Extraction from voacanga globosa
A total of 2.5 kg of air-dried V globosa leaves were used for extraction. The crude ethanolic extract was subsequently partitioned producing the hexane, aqueous and ethyl acetate partitions. Isocratic column FKURPDWRJUDSK\ FKORURIRUPPHWKDQRO RIWKHHWK\O
acetate partition produced nine column fractions (cf) as GHPRQVWUDWHG E\ 7/& DQDO\VLV 7KH ÀUVW IUDFWLRQ 9J
FI ZDV VHOHFWHG IRU IXUWKHU SXULÀFDWLRQ GXH WR LWV
observed bioactivity in the MTT assay (Table 1). Gradient HOXWLRQRI9JFIXVLQJDQHWK\ODFHWDWHKH[DQHVROYHQW
system yielded 11 gradient fractions. Gradient fraction, 9J*ZDVHOXWHGDWWKH HWK\ODFHWDWHKH[DQH
gradient mark and was determined to be the most bioactive gradient fraction (Table 1). HPLC analysis of Vg G10 produced three fractions: MP1, MP2 and MP3. Up to 9 mg of MP1, 2 mg of MP2 and 3 mg of MP3 were produced giving yields of 0.00035% for MP1, 0.00008% for MP2 and 0.00012% for MP3.
Methyl thiol tetrazolium (MTT) cytotoxicity assay Table 1 shows V globosa crude ethanolic extract with an average IC50RI ƫJP/DJDLQVWWKH+&7
cell line in the MTT assay. Solvent partitioning of the crude extract and subsequent MTT assays of the fractions, resulted to ethyl acetate and hexane fractions with average IC50DJDLQVW+&7RIƫJP/DQG
ƫJP/UHVSHFWLYHO\9JFIKDGWKHPRVWF\WRWR[LFPHDQ
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fractions. Gradient elution of Vg cf 1,2 produced Vg G10 fraction, which displayed the most cytotoxic mean IC50 RI ƫJP/ )UDFWLRQDWLRQ RI9J * WKURXJK
HPLC using a C18 semi-prep column produced three fractions (MP1, MP2 and MP3), MP1 (mean IC50 of 15.88 ƫJP/ 03 PHDQ,&50RIƫJP/ DQG
MP3 (mean IC50RIƫJP/ 03ZDVIXUWKHU
tested against the A549 cell line (mean IC50 RIƫJ
mL±0.51) and against the AA8 cell line (mean IC50 of 3.38 ƫJP/ 7DEOH
0 25.0 50.0 75.0 100.0
Newly diagnosed without treatment Newly diagnosed with treatment Persistence or recurrence Remission None Chemotherapy Radiotherapy Concurrent chemoradiation
Table 1.,&50 ƫJP/ RIWKHV globosa/HDI([WUDFW
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Extracts HCT116 A549 AA8 Doxorubicin 1.92±0.09 2.19±0.08 1.78±0.04 Ethanol 12.17±0.69
Ethyl Acetate 7.77±1.14 Vg cf 1,2 4.40±1.39 Vg G10 3.38±0.03
MP3 3.42±0.03 5.07±0.51 3.38±0.02
*Data were derived from three independent experiments with at least three replicates each. Data are presented here as means±SD
Table 2. Qualitative Analysis of Phytochemicals in V globosa([WUDFWV
Extracts Tannins Saponins Terpenoids Flavonoids Glycosides Phenolics Steroids Alkaloids
Ethanol (+) (+) (+) (-) (+) (-) (+) (-)
Hexane (+) (-) (+) (-) (+) (-) (+) (-)
Aqueous (-) (+) (-) (-) (-) (-) (-) (-)
Ethyl Acetate (+) (+) (+) (-) (-) (-) (+) (-)
Vg cf 1,2 (+) (+) (+) (-) (-) (-) (-) (-)
Vg G10 (+) (+) (+) (-) (-) (-) (-) (-)
Vg MP3 (-) (+) (+) - - - - -
*Presence of the constituent is represented by (+); absence is represented by (-); nt: not tested
Phytochemical screening
Qualitative analysis for phytochemicals revealed the presence of saponins and terpenoids in the MP3 fraction and all of its parent fractions (ethanolic crude extract, ethyl acetate partition, Vg cf 1,2 and Vg G10). Condensed tannins, cardiac glycosides and steroids were found to be present in the initial ethanolic crude extract but were removed in successive stages of extraction, being absent LQ WKH 9J 03 IUDFWLRQ 7HVWV IRU ÁDYRQRLGV SKHQROLF
compounds and alkaloids did not yield a positive result for all extracts tested (Table 2).
Assessment of cellular morphology
HCT116 cells incubated with the MP3 fraction at 3.42 ƫJP/ IRU KRXUV GHPRQVWUDWHG IHDWXUHV FRQVLVWHQW
with apoptosis which include membrane blebbing, cell shrinkage, surface detachment, loss of contact with neighboring cells and loss of spindle shape. These were also observed in those treated with doxorubicin (positive control) at its IC50RI ƫJP/EXWQRWLQ+&7FHOOV
treated only the DMSO (negative control). DMSO-treated cells demonstrated normal epithelia-like morphology, including spindle-shape, close attachment to neighboring cells and the substrate and were observed to grow in monolayers. Doxorubicin and MP3-treated cells also showed much reduced cell densities, implying cell death.
Diamidino-2-Phenylindole (DAPI) nucleic acid staining
,Q 03WUHDWHG FHOOV VLJQLÀFDQW LQFUHDVH S in apoptosis was observed at 24 hours of incubation with mean apoptotic indices of 0.238±0.083. The mean apoptotic indices for both treatment groups at 48 DQG KRXUV ZHUH IRXQG WR EH VLJQLÀFDQWO\ GLIIHUHQW
from the mean apoptotic indices of the DMSO-treated cells (negative control) at the same incubation periods (0.116±0.049 for 48 hours of incubation and 0.089±0.025 for 72 hours of incubation). At 12 hours of incubation, no VLJQLÀFDQWGLIIHUHQFHVZHUHREVHUYHGEHWZHHQWKHQHJDWLYH
(0.094±0.032) and positive controls (0.139±0.059) and the MP3-treated cells (0.150±0.061). HCT116 cells treated with MP3 and doxorubicin (positive control) displayed apoptotic bodies and condensed DNA indicating the occurrence of apoptosis.
Terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay
TUNEL assay screening of HCT116 cells treated with MP3 or doxorubicin (positive control) for 48, 60 and 72 hours demonstrated potent activity starting at the
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72 hours of incubation. Apoptosis was demonstrated by EULJKWJUHHQÁXRUHVFHQFHLQGLFDWLQJ'1$IUDJPHQWDWLRQ
At all incubation periods, mean apoptotic indices of MP3 DQGGR[RUXELFLQZHUHIRXQGWREHVLJQLÀFDQWO\GLIIHUHQW
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treated cells. However, apoptotic indices of MP3- and doxorubicin-treated cells showed no statistical difference H[FHSWDWKUZKHQ03VKRZHGVLJQLÀFDQWO\KLJKHU
apoptotic index (Figures 1 and 2 left).
JC-1 mitochondrial membrane potential assay
The MP3 fraction induced loss of mitochondrial membrane potential in HCT116 cells. Figure 2 left shows the collapse of mitochondrial membrane potential indicated E\FKDQJHVLQWKHUDWLRRIFHOOVZLWKUHGÁXRUHVFHQFH ZLWK
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collapsed mitochondrial membrane potentials). Cells incubated with the MP3 fraction for 72 hours at the IC50 RIƫJP/GHPRQVWUDWHGVLJQLÀFDQWO\ORZHU S UHGJUHHQÁXRUHVFHQFHUDWLRV PHDQÁXRUHVFHQFHUDWLRRI
1.208±0.155) compared to cells incubated with only the YHKLFOH'062 PHDQÁXRUHVFHQFHUDWLRRI
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of 1.180±0.134).
Discussion
Extracts of Voacanga globosa displayed toxicity against human cancer cell lines and are likely candidates )LJXUH781(/5HVXOWV OHIWZLWK$QWL%UG8PRXVH
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ULJKW ZLWK -& HCT116 Cells incubated for 72 hours with IC50 RI 03 $ % DW ƫJP/ '062 & ' DQG
doxorubicin (E, F) at IC50RI ƫJP/&HOOVZHUHYLHZHG
XQGHUÁXRUHVFHQFH $&( DQGEULJKWÀHOG %') DW;
PDJQLÀFDWLRQ
)LJXUH0HDQ$SRSWRWLF,QGLFHVRI+&7+XPDQ
&DUFLQRPD&HOOVDV$VVHVVHGE\781(/$VVD\Cells were treated with IC50 FRQFHQWUDWLRQVRI03DWƫJP/
doxorubicin (positive control) at IC50 ƫJP/ DQG WKH
vehicle solution DMSO (negative control). Apoptotic index was computed as the number of TUNEL-positive cells per replicate divided by the total number of cells. Values are means of three trials with three replicates each, ±SD.
Mean Apoptotic Index
Apoptosis-Induction by Voacanga globosa of Human Colon Carcinoma Cells for development as chemotherapeutic drugs. Previous
studies demonstrated that plant species related to V globosaSURGXFHFRPSRXQGVZLWKVLJQLÀFDQWDQWLFDQFHU
activities. Examples of these include the chemotherapeutic drugs vincristine and vinblastine which are derived from Catharanthus roseus belonging to the same family (Apocynaceae) as V globosa (Cragg and Newman, 2005).
Although many studies have demonstrated presence of bioactive compounds from the family Apocynaceae, very few studies have established bioactivity of compounds from Voacanga globosa. Here, the presence of anti-cancer substances in V globosaZDVFRQÀUPHGDQGGHPRQVWUDWHG
to have pro-apoptotic activity against HCT116 cells.
Anti-cancer activity of the V globosa extract is supported by results from the MTT assay when it showed potent cytotoxicity against the HCT116 human colon carcinoma cell line. According to the American National Cancer Institute (NCI), the standard for IC50 values LPSO\LQJVLJQLÀFDQWDFWLYLW\DJDLQVWFDQFHUFHOOOLQHVLV
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several other studies focusing on the induction of cell death in cancer cell lines through the MTT cytotoxicity assay -RNKDG]HHWDO &KDQJHWDO ,QWKHFXUUHQW
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in the cytotoxicity screening. The extract when pursued for further fractionation and bioassays demonstrated potent activity in the MTT assay according to this standard value with IC50 DV ORZ DV ƫJP/ ,QKLELWRU\ DFWLYLW\
of the extract against the HCT116 cells was observed at different stages of extraction, with generally increasing toxicity (decreasing IC50 ZLWKIXUWKHUSXULÀFDWLRQRIWKH
sample culminating in the MP3 fraction which displayed the most potent activity against HCT116 (average IC50 RIƫJP/ 7KH03IUDFWLRQEHFDXVHRILWVSRWHQW
activity against HCT116 cells compared to the MP1 and MP2 fractions, was selected for further assays.
The MP3 fraction has cytotoxic activity against the very sturdy A549 human lung carcinoma cell line and against the non-cancer AA8 Chinese hamster ovarian cell line. The low IC50 values of MP3 against the +&7$ DQG$$ FHOO OLQHV ƫJP/ DJDLQ
indicate an ability to inhibit cell growth even at very low concentrations.
MP3 demonstrated an ability to induce time-dependent changes in the nuclear morphology of HCT116 cells at
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changes in nuclear and cell morphology culminate in the fragmentation of cells into membrane-bounded bodies (apoptotic bodies) which has been cited previously as a hallmark feature for apoptosis (Savill and Fadok, 2000;
Kurosaka et al., 2003; Wu et al., 2013). Pro-apoptotic activity is also demonstrated in the changes in cellular morphology after 72 hours of incubation, with cells displaying loss of spindle shape, membrane blebbing, surface detachment and the formation of cellular granules.
Cytotoxicity of MP3 due to the induction of apoptosis in HCT116 is supported by results with the TUNEL assay.
DNA double strand breaks due to extensive fragmentation of nuclear DNA in karyorrhexis is a distinguishing feature
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.UHVVHODQG*URVFXUWK 'DU]\QNLHZLF]HWDO
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cells undergoing cell death and is a common assay in DSRSWRVLVUHVHDUFK 0D]DKHU\HWDO *RKDQG.DGLU
2011; Hasan et al., 2011; Tan et al., 2013; Wu et al., 2013).
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fragmentation from 48 to 72 hours of incubation.
MP3 demonstrates significant ability to induce PLWRFKRQGULDO PHPEUDQH GHSRODUL]DWLRQ DV DVVHVVHG
by the JC-1 mitochondrial membrane potential assay.
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form aggregates within viable mitochondria leading to UHG ÁXRUHVFHQFH RU LQ WKH LQVWDQFH RI PLWRFKRQGULDO
PHPEUDQHSRWHQWLDOGHVWDELOL]DWLRQORFDOL]HWKURXJKRXW
WKHF\WRVRO\LHOGLQJJUHHQÁXRUHVFHQFH&ROODSVHRIWKH
mitochondrial potential is brought about by the formation of the mitochondrial permeability transition (MPT) pore in the mitochondrial membrane which facilitates discharge of pro-apoptotic factors from the intermembrane space into the cytosol (Saelens et al., 2004; Brenner and Moulin, 2012). Studies have demonstrated that collapse of the mitochondrial membrane potential due to opening of the MPT pore typically triggers apoptosis (Qanungo et al., 2005; Gao et al., 2013; Ramkumar et al., 2013). The loss of mitochondrial membrane potential in MP3-treated cells supports the observed cytotoxic activity of the fraction in the MTT assay. The MTT assay is based on the ability of YLDEOHFHOOVWRUHGXFH077LQWRIRUPD]DQDQGWKLVUHDFWLRQ
occurs in the mitochondria. The use of JC-1 demonstrates that the mitochondria of MP3-treated HCT116 cells have been compromised hence, unable to reduce MTT leading to the observed toxicity in the MTT assay. The cytotoxic activity of the fraction may thus be facilitated by the LQGXFWLRQRIPLWRFKRQGULDOPHPEUDQHGHSRODUL]DWLRQ
The ability of MP3 to induce apoptosis is advantageous in the treatment of cancer. Unlike in necrosis, pro- LQÁDPPDWRU\F\WRVROLFFRQWHQWVDUHQRWUHOHDVHGEXWDUH
kept inside vesicles which are eventually phagocytosed when phagocytic markers are exposed during the terminal steps of apoptosis. The induction of apoptosis thereby DYRLGVLQÁDPPDWLRQDQGWLVVXHGDPDJH 6DYLOODQG)DGRN
2000; Kurosaka et al., 2003; Ravichandran 2011) thus reducing incidence of negative side effects on surrounding normal tissue.
Phytochemical analysis of the parent fractions of MP3 demonstrated the presence of several known bioactive FRPSRXQGJURXSV)XUWKHUVFUHHQLQJFRQÀUPHGVDSRQLQV
and terpenoids to be present in the MP3 fraction. These may likely be the cause of the observed cytotoxicity as these substances have all been previously shown to possess anticancer activity (Polo and de Bravo, 2006; Tin et al., 2007; Lu et al., 2011; Thoppil and Bishayee, 2011).
In conclusion, this study demonstrated that the leaves of V globosa are possible sources of anti-cancer compounds validating its use by indigenous people in Bataan, Philippines as antitumor agents. Further fractionation of the extract produced successively more cytotoxic fractions culminating in the MP3 fraction. MP3 was demonstrated to have toxicity to A549 human lung carcinoma and the AA8 Chinese hamster ovarian cell lines,
indicating that the observed cytotoxicity is not unique WR +&7 EXW WR RWKHU FHOO OLQHV DV ZHOO 6LJQLÀFDQW
cytotoxic activity against A549 demonstrates an ability to resist drug resistance mechanisms present in the A549 cells. The MP3 fraction was also demonstrated through DAPI staining and TUNEL assay, to induce apoptosis due to a reduction of the mitochondrial membrane potential determined by treatment with JC-1. Cytotoxic and apoptosis-inducing activities of the MP3 fraction are likely to have been caused by saponins or terpenoids.
Acknowledgements
We gratefully acknowledge the research grants SURYLGHGE\WKH8QLYHUVLW\RIWKH3KLOLSSLQHV2IÀFHRI
the Vice Chancellor for Research and Development (UP OVCRD), University of the Philippines, Natural Sciences Research Institute (UPNSRI) and the Department of Science and Technology-Philippine Council for Health Research and Development (DOST-PCHRD). We acknowledge further, the Institute of Biology and the Institute of Chemistry, University of the Philippines, Diliman for the facilities and work space where this research was done.
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